Cheng AM et al. (2000), The lefty-related factor Xatv acts as a feedbac...

XB-ART-11539

Development 2000 Mar 01;1275:1049-61. doi: 10.1242/dev.127.5.1049.

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The lefty-related factor Xatv acts as a feedback inhibitor of nodal signaling in mesoderm induction and L-R axis development in xenopus.

Cheng AM , Thisse B , Thisse C , Wright CV .

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In mouse, lefty genes play critical roles in the left-right (L-R) axis determination pathway. Here, we characterize the Xenopus lefty-related factor antivin (Xatv). Xatv expression is first observed in the marginal zone early during gastrulation, later becoming restricted to axial tissues. During tailbud stages, axial expression resolves to the neural tube floorplate, hypochord, and (transiently) the notochord anlage, and is joined by dynamic expression in the left lateral plate mesoderm (LPM) and left dorsal endoderm. An emerging paradigm in embryonic patterning is that secreted antagonists regulate the activity of intercellular signaling factors, thereby modulating cell fate specification. Xatv expression is rapidly induced by dorsoanterior-type mesoderm inducers such as activin or Xnr2. Xatv is not an inducer itself, but antagonizes both Xnr2 and activin. Together with its expression pattern, this suggests that Xatv functions during gastrulation in a negative feedback loop with Xnrs to affect the amount and/or character of mesoderm induced. Our data also provide insights into the way that lefty/nodal signals interact in the initiation of differential L-R morphogenesis. Right-sided misexpression of Xnr1 (endogenously expressed in the left LPM) induces bilateral Xatv expression. Left-sided Xatv overexpression suppresses Xnr1/XPitx2 expression in the left LPM, and leads to severely disturbed visceral asymmetry, suggesting that active 'left' signals are critical for L-R axis determination in frog embryos. We propose that the induction of lefty/Xatv in the left LPM by nodal/Xnr1 provides an efficient self-regulating mechanism to downregulate nodal/Xnr1 expression and ensure a transient 'left' signal within the embryo.

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Species referenced: Xenopus
Genes referenced: cer1 chrd gsc lefty nodal nodal1 nodal2 nog pitx2 tbxt tgfb1 tnni3

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	Fig. 1. Structure and expression of Xatv. (A) Xatv is 66%, 37% and 35% identical to zebrafish atv, mouse lefty-1 and lefty-2, respectively (overline indicates hydrophobic signal sequence; gray boxes, putative proteolytic cleavage sites; dashes, identical residues; dots, alignment gaps). Asterisks indicate cysteines of the TGFb ligand ‘cysteine knot’; additional cysteines residues conserved between lefty/atv factors are indicated. (B) TGFb/BMP, lefty, and Xatv pre-proproteins. Gray, black and white boxes indicate signal sequence, pro region and mature ligand peptide, respectively. (C) RT-PCR analysis of Xatv expression during Xenopus embryogenesis (stages indicated; 0.1 embryoequivalents/ lane). FGFR, loading control; MBT, mid-blastula transition.
	Fig. 2. Marginal zone and midline expression of Xatv. (A) Stage 10 embryo, dorsal view. Xatv transcripts are enriched dorsally in the marginal zone. (B-D) Stage 10.5, 11 and 12. Dorsovegetal views, dorsal up. Xatv expression within the forming dorsal midline. Arrowhead, dorsal lip; asterisk, yolk plug. (E) Stage 12, longitudinal section, approximate location shown in D. Xatv expression is seen in posterior neuroectoderm (open arrow), more anterior mesendoderm (solid arrow), and at lower levels in newly involuting mesendoderm (open arrowhead). Inset: higher magnification of posterior midline; Xatv is mostly expressed in deep neuroectoderm (bracket; dorsal lip indicated). (F) Stage 14. Midline expression, dorsal view, anterior left. Bracket shows the level of the section in G (arrowheads, circumblastoporal expression). (G) Stage 14, transverse section: Xatv expression in the prospective floorplate and underlying mesendoderm. (H) A more posterior section than in G shows Xatv expression only within the prospective floorplate. (I) Stage 17. Dorsal view, anterior left. (J) Stage 19. Dorsal view, anterior left. Inset: magnification of midline expression with transient notochord expression bracketed. (K,L) Stage 23 lateral view and stage 25 dorsal view, respectively (anterior left), showing Xatv expression in left dorsal endoderm (open arrowheads; see P for section), bilateral expression in the posterior dorsal endoderm (open arrows), and left LPM (solid arrowhead). (M) Stage 23. An anterior transverse section; Xatv expression in floorplate and hypochord. (N) Stage 25. (O) Stage 29/30. (P) Stage 25. Arrow, left dorsal endoderm expression. a, archenteron; b, blastocoel; fp, floorplate; n, notochord; h, hypochord.
	Fig. 3. Asymmetric Xatv expression. (A-D) Uncleared embryos, (F-I) cleared embryos (stages indicated); lateral views, anterior left. Solid arrowheads, Xatv expression in left LPM; open arrowheads, Xatv expression in left dorsal endoderm. (E) Transverse section of stage 25 embryo; approximate location indicated in C. Note expression in floorplate and hypochord. Solid arrowhead, Xatv expression in the left LPM. (J,K) Stage 32. Sagittal and transverse section, respectively, of heart regions of an embryo double labeled for cardiac troponin I (pink) and Xatv (purple), showing Xatv expression in the looping heart tube (bracket). *sectioning artifact. Epidermal coloration is normal embryo pigmentation. c, cement gland; fp, floorplate; h, hypochord.
	Fig. 4. Inhibition of Xnr2 and activin activity by Xatv. Animal caps injected with Xatv plus or minus Xnr2 or activin RNA (pg/embryo indicated) were assayed at stage 10.5 or stage 24. At 1:1 ratios (10 pg each RNA), marker induction was similar to that caused by Xnr2 alone. In contrast, a 10:1 mix of Xatv:Xnr2 RNA suppressed organizer-specific markers (cerberus, chordin, goosecoid and noggin). At 5:1 or 50:1 ratios of Xatv:activin RNA, Xatv suppressed all markers tested.
	Fig. 5. Induction of Xatv in the LPM by Xnr1. pXEX/Xnr1 or pCSKA/Xnr1 was injected into the right side of 4-cell embryos to drive Xnr1 expression from late blastula or gastrula stages, respectively, and Xatv expression analyzed at stage 24-25. Dorsal views, anterior up. (A) Normal asymmetric expression of Xatv; (B) bilateral expression; (C) right side expression and (D) no expression. Bilateral expression predominated (Table 1). (E) Animal caps injected with Xnr1, Xnr2, or activin RNA were assayed at stage 10.5 for Xatv and Xbra. Xbra, a pan-mesodermal marker, measured induction efficiency. FGFR, loading control.
	Fig. 6. Suppression of left LPM Xnr1 and XPitx2 expression by Xatv. pXEX/Xatv or pCSKA/Xatv was injected into the left side of 4-cell embryos and (A-D) XPitx2, or (E,F) Xnr1 expression analyzed. (A,B) Lateral views and (C,D) dorsal views (anterior left). (A,C) Normal XPitx2 expression in left LPM (bracket) is (B,D) suppressed by left-sided Xatv overexpression. Bilateral XPitx2 expression in the eyes, head, branchial arches and cement gland (arrowheads in A,B) remains relatively unaffected. (E,F) Dorsal views, anterior up. (E) Normal Xnr1 expression in left LPM is (F) suppressed by left-sided Xatv overexpression.
	Fig. 7. Morphological analysis of Xatv-injected embryos. Plasmids (100 pg) encoding Xnr1 or Xatv were injected into either the right (R) or left (L) of 4-cell embryos and morphology analyzed at stage 43-45. (A,D,G,J,M) Lateral views; (B,E,H,K,N) corresponding ventral views; anterior up. (C,F,I,L,O) Higher magnification of heart region. (A-C) Uninjected embryo. (D-F) Reversed heart and gut situs in right-side Xnr1 injections, for comparative purposes. (G-I) Normal heart and gut situs in right-side Xatv-injected embryos. (J-O) Two examples of embryos receiving Xatv on the left (percentage incidences indicated; see Table 4 for details).
	Fig. 8. A model for the role of Xatv in the establishment of L-R asymmetry. Time flows from top to bottom. A hypothetical factor ‘X’ that is proposed to propagate node-proximate asymmetries activates Xnr1 within the left LPM during early tailbud stages. Xnr1 activates XPitx2 expression directly, or through intermediary steps. Xnr1 also induces asymmetric Xatv expression. With a delay period, Xatv acts in a negative feedback loop to suppress Xnr1 expression. Preliminary data suggest that Xnr1 regulates its own expression. In the experiments reported here, Xatv may inhibit XPitx2 directly (dashed bar), or perhaps more likely by directly suppressing Xnr1 (solid bar). The possibility of yet undiscovered genes in Xenopus acting in a right-sided gene cascade, as in other species (see text), is indicated.